Cholera toxin (CT), an etiologic agent in cholera, exerts its effect on cells through the ADP-ribosylation of guanine nucleotide-binding (G) proteins that are critical for signaling from cell surface receptors to their intracellular targets (e.g., adenylyl cyclase). The toxin consists of one A subunit, which is an ADP-ribosyltransferase and five B subunits, which bind the toxin to cell surface ganglioside GM1. The enzymatic activity of the A subunit is latent, and activity requires proteolysis and reduction of a single disulfide resulting in the formation of a catalytically active A1 protein and a much smaller A2 protein derived from the carboxy terminus of the A subunit. The activity of the A1 protein is enhanced by 20 kDa ADP-ribosylation factors or ARFs, in a GTP-dependent manner. (1) To determine if the latent form of the toxin expresses an ARF activation site, an inactive mutant form of E. coli heat-labile enterotoxin (LT), with a critical glutamate to lysine substitution at position 112 of the A subunit, was utilized as a competitor in a cholera toxin assay containing limiting ARF. The holotoxin did not significantly inhibit the ARF-stimulated ADP-ribosyltransferase activity of CT A subunit. Trypsinization and reduction of LT resulted in a potent inhibitor; reduction alone did not. Trypsinized LT did not inhibit ARF stimulation of a purified, alkylated CTA1 protein, which is not dependent on reduction for activity. These studies are consistent with the hypothesis that the ARF site on CT is not accessible in the latent toxin. Release of the CT A1 protein, which is necessary for expression of ADP-ribosyltransferase activity, is also required for appearance of the ARF binding site. (2) The cholera toxin A1 protein ADP-ribosylates arginine residues in G proteins. Mammalian cells contain enzymes, termed ADP-ribosylarginine hydrolases, that cleave the ADP-ribosyl-arginine bond, releasing free arginine and could play a role in recovery from cholera. To examine the structural features and conservation of hydrolases, they were cloned from rat, mouse and human sources. The hydrolases from these species exhibited considerable amino acid identity.